Robust Multichannel Encoding for Highly Multiplexed Quantitative PCR.

The gold standard of molecular pathogen detection is the quantitative polymerase chain reaction (qPCR). Modern qPCR instruments are capable of detecting 4-6 analytes in a single sample: one per optical detection channel. However, many clinical applications require multiplexing beyond this traditional single-well capacity, including the task of simultaneously testing for SARS-CoV-2 and other respiratory pathogens. This can be addressed by dividing a sample across multiple wells, or using technologies such as genomic sequencing and spatial arrays, but at the expense of significantly higher cost and lower throughput compared with single-well qPCR. These trade-offs represent unacceptable compromises in high-throughput screening scenarios such as SARS-CoV-2 testing. We demonstrate a novel method of detecting up to 20 targets per well with standard qPCR instrumentation: high-definition PCR (HDPCR). HDPCR combines TaqMan chemistry and familiar workflows with robust encoding to enable far higher levels of multiplexing on a traditional qPCR system without an increase in cost or reduction in throughput. We utilize HDPCR with a custom 20-Plex assay, an 8-Plex assay using unmodified predesigned single-plex assays from Integrated DNA Technologies and a 9-Plex pathogen panel inclusive of SARS-CoV-2 and other common respiratory viruses. All three assays were successful when tested on a variety of samples, with overall sample accuracies of 98.8, 98.3, and 100%, respectively. The HDPCR technology enables the large install base of qPCR instrumentation to perform mid-density multiplex diagnostics without modification to instrumentation or workflow, meeting the urgent need for increased diagnostic yield at an affordable price without sacrificing assay performance.

Rare LRP6 variants identified in spina bifida patients.

Several single-nucleotide variants (SNVs) in low-density lipoprotein receptor-related protein 6 (Lrp6) cause neural tube defects (NTDs) in mice. We therefore examined LRP6 in 192 unrelated infants from California with the NTD, spina bifida, and found four heterozygous missense SNVs, three of which were predicted to be deleterious, among NTD cases and not in 190 ethnically matched nonmalformed controls. Parents and siblings could not be tested because of the study design. Like Crooked tail and Ringleschwanz mouse variants, the p.Tyr544Cys Lrp6 protein failed to bind the chaperone protein mesoderm development and impaired Lrp6 subcellular localization to the plasma membrane of MDCK II cells. Only the p.Tyr544Cys Lrp6 variant downregulated canonical Wnt signaling in a TopFlash luciferase reporter in vitro assay. In contrast, three Lrp6 mutants (p.Ala3Val, p.Tyr544Cys, and p.Arg1574Leu) increased noncanonical Wnt/planar cell polarity (PCP) signaling in an Ap1-luciferase assay. Thus, LRP6 variants outside of YWTD repeats could potentially predispose embryos to NTDs, whereas Lrp6 modulation of Wnt/PCP signaling would be more essential than its canonical pathway role in neural tube closure.

Brief report novel mechanism for valproate-induced teratogenicity.

BACKGROUND: Valproic acid (VPA) is a commonly prescribed drug for those affected by epilepsy and bipolar disorders. VPA has a well known teratogenic potential, causing a variety of birth defects including neural tube defects (NTDs) and other congenital malformations, when women are treated with this medication during pregnancy. Unfortunately, the mechanism by which VPA is teratogenic remains unknown, although a range of potential mechanisms including histone deacetylase inhibition and folate antagonism have been proposed. The latter is of considerable importance, as clinicians need to know if additional folate supplements can prevent VPA-induced defects. METHODS: We herein approach this question experimentally, using enzyme-linked immunosorbent assay assays and cell culture modeling, to demonstrate that VPA serves as a noncompetitive inhibitor of the high affinity folate receptors. RESULTS: Binding affinities experimentally determined through enzyme-linked immunosorbent assay assays indicate that VPA serves as a noncompetitive substrate that can lessen the ability of the three primary folate forms to bind to the high affinity folate receptors. Tests in HEK293T cells indicate that the membrane-bound folate receptors of VPA treated cells bind significantly lower amounts of folic acid than do untreated cells. CONCLUSION: If these data translate to the overall transport and subsequent bioavailability of folates, noncompetitive inhibition of the folate receptors by VPA may serve to lower the bioavailable folates in VPA treated mothers. This represents a novel mechanism by which in utero VPA exposure could be disrupting developmental processes by noncompetitively binding to the folate receptors during embryogenesis, thus inducing the wide range of defects seen in babies born to VPA treated mothers.

Neural tube defects, folate, and immune modulation.

Periconceptional supplementation with folic acid has led to a significant worldwide reduction in the incidence of neural tube defects (NTDs). However, despite increasing awareness of the benefits of folic acid supplementation and the implementation of food fortification programs in many countries, NTDs continue to be a leading cause of perinatal morbidity and mortality worldwide. Furthermore, there exists a significant subgroup of women who appear to be resistant to the protective effects of folic acid supplementation. The following review addresses emerging clinical and experimental evidence for a role of the immune system in the etiopathogenesis of NTDs, with the aim of developing novel preventative strategies to further reduce the incidence of NTD-affected pregnancies. In particular, recent studies demonstrating novel roles and interactions between innate immune factors such as the complement cascade, neurulation, and folate metabolism are explored.

Challenges and Future Prospects for the Delivery of Biologics: Oral Mucosal, Pulmonary, and Transdermal Routes.

Biologic products are large molecules such as proteins, peptides, nucleic acids, etc., which have already produced many new drugs for clinical use in the last decades. Due to the inherent challenges faced by biologics after oral administration (e.g., acidic stomach pH, digestive enzymes, and limited permeation through the gastrointestinal tract), several alternative routes of administration have been investigated to enable sufficient drug absorption into systemic circulation. This review describes the buccal, sublingual, pulmonary, and transdermal routes of administration for biologics with relevant details of the respective barriers. While all these routes avoid transit through the gastrointestinal tract, each has its own strengths and weaknesses that may be optimal for specific classes of compounds. Buccal and sublingual delivery enable rapid drug uptake through a relatively permeable barrier but are limited by small epithelial surface area, stratified epithelia, and the practical complexities of maintaining a drug delivery system in the mouth. Pulmonary delivery accesses the highly permeable and large surface area of the alveolar epithelium but must overcome the complexities of safe and effective delivery to the alveoli deep in the lung. Transdermal delivery offers convenient access to the body for extended-release delivery via the skin surface but requires the use of novel devices and formulations to overcome the skin's formidable stratum corneum barrier. New technologies and strategies advanced to overcome these challenges are reviewed, and critical views in future developments of each route are given.

Inhaled Biologics: From Preclinical to Product Approval.

BACKGROUND: Delivery of pharmacologically active compounds to the lung for systemic effects is well known and recently has entered a new era with several products achieving regulatory approval. This review focuses on the barriers to pulmonary delivery of biologics. METHODS: Lessons learned from the development of recently approved products will be reviewed to shed light on the current challenges that are faced when developing biological products for inhaled delivery. RESULTS: The text and tables presented herein consolidate the current data and ongoing research regarding biological, inhaled products. CONCLUSION: With this basis, we also review the future prospects for pulmonary delivery of biologics for systemic delivery and how the biological and physical barriers may be overcome.

The application of a chemical determination of N-homocysteinylation levels in developing mouse embryos: implication for folate responsive birth defects.

Elevated homocysteine levels have long been associated with various disease states, including cardiovascular disease and birth defects, including neural tube defects (NTDs). One hypothesis regarding the strong correlation between these various disorders and high levels of homocysteine is that a reactive form of this small molecule can attach to mammalian proteins in a phenomenon known as homocysteinylation. These posttranslational modifications may become antigenic or may even directly disrupt certain protein function. It remains to be determined whether dietary influences that can cause globally increased levels of circulating homocysteine confer negative effects maternally, or may otherwise negatively and materially impact the metabolic balance in developing embryos. Herein we present the application of a chemical method of determination of N-homocysteinylation to a set of neural tube closure stage mouse embryos and their mothers. We explore the uses of this newly described technique to investigate levels of maternal and embryonic N-homocysteinylation using dietary manipulations of one-carbon metabolism with two known folate-responsive NTD mouse models. The data presented reveal that although diet appeared to have significant effects on the maternal metabolic status, those effects did not directly correlate to the embryonic folate or N-homocysteinylation status. Our studies indicate that maternal diet and embryonic genotype most significantly affected the embryonic developmental outcome.

Amorphous or Crystalline? A Comparison of Particle Engineering Methods and Selection.

This review is intended to provide a critical account of the current goals and technologies of particle engineering regarding the production of crystalline and amorphous particles. The technologies discussed here cover traditional crystallization technologies, supercritical fluid technologies, spray drying, controlled solvent crystallization, and sonocrystallization. Also recent advancements in particle engineering including spray freezing into liquid, thin-film freeze-drying, PRINT technology are presented. The paper also examines the merits and limitations of these technologies with respect to their methods of characterization. Additionally a section discussing the utility of creating amorphous and crystalline formulation approaches in regards to bioavailability and utility in formulation is presented.

Delivery and therapeutic applications of gene editing technologies ZFNs, TALENs, and CRISPR/Cas9.

In recent years, several new genome editing technologies have been developed. Of these the zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 RNA-guided endonuclease system are the most widely described. Each of these technologies utilizes restriction enzymes to introduce a DNA double stranded break at a targeted location with the guide of homologous binding proteins or RNA. Such targeting is viewed as a significant advancement compared to current gene therapy methods that lack such specificity. Proof-of-concept studies have been performed to treat multiple disorders, including in vivo experiments in mammals and even early phase human trials. Careful consideration and investigation of delivery strategies will be required so that the therapeutic potential for gene editing is achieved. In this review, the mechanisms of each of these gene editing technologies and evidence of therapeutic potential will be briefly described and a comprehensive list of past studies will be provided. The pharmaceutical approaches of each of these technologies are discussed along with the current delivery obstacles. The topics and information reviewed herein provide an outline of the groundbreaking research that is being performed, but also highlights the potential for progress yet to be made using these gene editing technologies.

Identification of a redox-modulatory interaction between uncoupling protein 3 and thioredoxin 2 in the mitochondrial intermembrane space.

UNLABELLED: Uncoupling protein 3 (UCP3) is a member of the mitochondrial solute carrier superfamily that is enriched in skeletal muscle and controls mitochondrial reactive oxygen species (ROS) production, but the mechanisms underlying this function are unclear. AIMS: The goal of this work focused on the identification of mechanisms underlying UCP3 functions. RESULTS: Here we report that the N-terminal, intermembrane space (IMS)-localized hydrophilic domain of mouse UCP3 interacts with the N-terminal mitochondrial targeting signal of thioredoxin 2 (Trx2), a mitochondrial thiol reductase. Cellular immunoprecipitation and in vitro pull-down assays show that the UCP3-Trx2 complex forms directly, and that the Trx2 N-terminus is both necessary and sufficient to confer UCP3 binding. Mutation studies show that neither a catalytically inactivated Trx2 mutant, nor a mutant Trx2 bearing the N-terminal targeting sequence of cytochrome c oxidase (COXMTS-Trx2) bind UCP3. Biochemical analyses using permeabilized mitochondria, and live cell experiments using bimolecular fluorescence complementation show that the UCP3-Trx2 complex forms specifically in the IMS. Finally, studies in C2C12 myocytes stably overexpressing UCP3 (2.5-fold) and subjected to Trx2 knockdown show that Trx2 is required for the UCP3-dependent mitigation of complex III-driven mitochondrial ROS generation. UCP3 expression was increased in mice fed a high fat diet, leading to increased localization of Trx2 to the IMS. UCP3 overexpression also increased expression of the glucose transporter GLUT4 in a Trx2-dependent fashion. INNOVATION: This is the first report of a mitochondrial protein-protein interaction with UCP3 and the first demonstration that UCP3 binds directly, and in cells and tissues with mitochondrial thioredoxin 2. CONCLUSION: These studies identify a novel UCP3-Trx2 complex, a novel submitochondrial localization of Trx2, and a mechanism underlying UCP3-regulated mitochondrial ROS production.

Infrared spectroscopy of solid hydrogen sulfide and deuterium sulfide.

The infrared spectra of solid hydrogen sulfide (H2S) and deuterium sulfide (D2S) were collected at very low temperatures. Vapor deposition of thin films at the lowest temperature of 10 K produced amorphous solids while deposition at 70 K yielded the crystalline phase III. Infrared interference fringe patterns produced by the films during deposition were used to determine the film thickness. Careful measurement of the integrated absorbance peaks, along with the film thickness, allowed determination of the integrated band intensities. This report represents the first complete presentation of the infrared spectra of the amorphous solids. Observations of peaks near 3.915 and 1.982 microm (ca. 2554 and 5045 cm(-1), respectively) may be helpful in the conclusive identification of solid hydrogen sulfide on the surface of Io, a moon of Jupiter.